Klapperich Lab:Collagen-GAG Meshes

Overview

Protocol for making collagen-GAG meshes (150 ML SAMPLES).

Materials

Reagents

• 250 mL of 0.05M acetic acid
• 0.75 g of collagen (Type I, Insoluble from Bovine Achilles Tendon)
• 0.0795 g chondroitin-6-sulfate

Equipment

• Homogenizer
• 250 ml jacketed beaker
• Cold water bath
• Ultrasonicator
• Magnetic stir plate
• Lyophilizer
• Vacuum oven
• Metal mold

Procedure

Making 0.05 M solution of acetic acid

Note: This solution has a shelf life of one week.

1. Fill sterilized bottle with 250 mL of Millipore water.
2. Add 715 µL acetic acid.

0.5% w/v Dispersion of Collagen in Acetic Acid

Note: Keep collagen mixture at below 20°C.

1. Add 0.75 g of collagen (Type I, Insoluble from Bovine Achilles Tendon) to a 250 ml jacketed beaker beaker. (0.5 % w/v, this type of collagen should be stronger than from calf skin)
2. Add 150 ml of 0.05 M acetic acid to the beaker.
3. Mix solution with homogenizer at full speed for approximately 5 minutes, or until no large fibrils or chunks are apparent. Make sure that tip has been washed thoroughly (ethanol, rinse with Millipore water)
4. Connect the beaker inlet and outlet to the bath (Fisher Scientific), and set bath temperature at 4°C. Turn on bath and allow the bath to reach at least 10°C. Make sure bath is filled to second line. Add a few drops of Algicide each time the bath is used.
5. Set the sonicater controller to the ID03 program. (Amplitude 100% (dial), 59 min, 5 sec on, 10 sec off). Press RECALL. Type in 03 and press ENTER.
6. Place sonicater horn in collar, making sure tip of sonicater does not touch the bottom of the beaker. Press START/STOP on sonicater controller.

Addition of the 0.53 % w/v Solution of Chondroitin 6-Sulfate (C6S) in Acetic Acid

1. Measure 0.0795 g C6S into a 50 ml polypropylene centrifuge tube.
2. Add 15 ml 0.05 M acetic acid to the tube. This should be 10% of the volume of the collagen mixture.
3. Mix the C6S solution by shaking with cap on.
4. Transfer collagen solution to a sterilized non-jacketed beaker. Add clean stir bar and place on stir plate at setting 5.
5. Add C6S solution dropwise to stirring collagen solution. Roughly 1 ml/min addition.
6. Remove stir bar and transfer collagen-GAG solution back to jacketed beaker.
7. Continue sonicating at the same settings for 90 more minutes to ensure an even dispersion.
8. Degas mixture in a vacuum flask for 30 minutes until bubbles are no longer present.

Setting the Scaffold (for any initial volume)

1. Pour 4 mL collagen-C6S mixture into each well in a 25-well metal mold.
2. Freeze at -20°C for 15 minutes, and then transfer to -80°C for one day.
3. Transfer to sterilized lyophilizer for 2 days.
4. Heat in vacuum oven at 105°C, at a vacuum reading of ~22 inHg, for 2 days in order for the meshes to be sturdier.
5. Keep meshes on dessicant to keep dry.

Notes

• The mixture may be stored in a capped bottle at 4°C for about 4 months.
• Re-blend at 4°C at an amplitude of 60 with the pulser set to 5 sec for 15 minutes to use.

References

Relevant papers and books

1. Pieper JS, Oosterhof A, Dijkstra PJ, Veerkamp JH, and van Kuppevelt TH. Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate. Biomaterials. 1999 May;20(9):847-58. DOI:10.1016/s0142-9612(98)00240-3 |

Contact

• Cass